Current methods of screening for drug resistance involve isolation and culturing of microorganisms from clinical specimens. When a sufficient culture is obtained, the isolated microorganisms are tested for sensitivity or resistance to a drug by their ability to grow in the presence of the drug (B. E. Murray (1990) Clinical Microbiology Reviews 3: 46-65). Growth of the microorganisms in the presence of a drug is indicative of resistance to that drug.
Such prior art methods are unsatisfactory in several respects. First, many significant pathogens are difficult to isolate from clinical specimens and/or difficult to culture in sufficient quantity to perform the standard drug resistance screening tests. In addition, such procedures can require several days since the microorganisms must first be cultured to obtain a sufficient number of cells, and then must be cultured again in the presence of the desired drugs to obtain a result. Often, the microorganisms must also be identified biochemically. A patient, particularly if marginally effective drug therapy has initially been instituted on an empirical basis, can deteriorate significantly while awaiting the results of such drug resistance screening. Further, the standard methods are relatively insensitive in detecting resistance due to their reliance on visible culture growth during the period of the assay. These methods are therefore only capable of demonstrating the existence of drug resistant organisms when the proportion of such microorganisms in the culture is sufficiently high to have a significant effect on visible culture growth. As a result, microorganisms are often erroneously characterized as susceptible to a drug, with potentially harmful consequences for the patient (See J. C. Pechere (1989) Eur. J. Cancer Clin. Oncol. 25: S17-S23, Supplement 2).
Microorganisms have developed a variety of mechanisms of resistance to drugs. These include, in part, the acquisition of genes which produce an enzyme capable of breaking down a particular drug (e.g., .beta.-lactamases, conferring resistance to .beta.-lactam antibiotics such as penicillin), cell membrane mutations which decrease permeation of the drug into the cell (e.g., altered membrane porins which confer resistance to quinolone antibiotics), and alterations in molecules which are the targets of the drugs so that interaction of the drug with its target is reduced (e.g., mutations in DNA gyrase conferring resistance to quinolones; alterations in penicillin-binding proteins providing resistance to .beta.-lactam antibiotics).
Each of the many mechanisms of drug resistance is, ultimately, the result of genetic alteration which confers the resistant phenotype. Under the selective pressure applied by exposure to the drug, rare resistant microorganisms which arise in a sensitive population are able to survive and multiply, thus increasing their relative proportion compared to sensitive microorganisms. The genetic alteration or mutation can involve the acquisition of a plasmid or transposable element carrying a new gene which confers resistance, or it can involve mutation of a gene (usually chromosomal) coding for a normal component of the cell. Particularly in the case of plasmids and transposable elements, the acquired drug resistance can then be transferred to other microorganisms, either of the same species or of different species. As a result of such resistance transfer, there is often significant homology between the genes conferring resistance to a drug in different species of microorganisms.
For example, resistance to .beta.-lactam antibiotics is known to be acquired by at least two genetic mechanisms: 1) acquisition of a gene coding for .beta.-lactamase, and 2) alteration of one or more of the penicillin binding proteins in the cell membrane (J. C. Pechere (1989) Eur. J. Cancer Clin. Oncol. 25: S17-S22; R. Fontana et al. (1990) Eur. J. Clin. Microbiol. Infect. Dis. 9: 103-105; A. R. Wanger et al. (1990) The Journal of Infectious Diseases 161: 54-58; C. E. Nord (1990) Reviews of Infectious Diseases 12: S231-S234).
Resistance to the quinolones is also known to be mediated by several genetic mechanisms, including: 1) mutations in the genes coding for either of the two subunits of DNA gyrase (the gyrA and gyrB genes), 2) mutations in one or more genes for the outer membrane porins, and 3) altered outer membrane lipopolysaccharides (LPS) (D. C. Hooper et al. (1989) The American Journal of Medicine 87: 6C-17S-6C-23S; J. S. Wolfson (1989) Eur. J. Clin. Microbiol. Infect. Dis. 8: 1080-1092; C. S. Lewin et al. (1990) J. Med. Microbiol. 31: 153-161; J. S. Wolfson et al. (1989) Reviews of Infectious Diseases 11: S960-S978).
Macrolide-lincosamide-streptogramin B resistance, which includes resistance to Clindamycin and Erythromycin, is mediated by acquisition of a gene for an RNA methylase which methylates 23S rRNA so that the drug is no longer bound. These genes (ermF and related sequences) are highly homologous in all of the bacterial species tested, although they can exist either on plasmids or as chromosomal elements (M. Halula et al. (1990) Reviews of Infectious Diseases 12: S235-S242). The ermF like sequences are often associated with a genetic element conferring resistance to tetracycline (tetF).
The polymerase chain reaction ("PCR") has been a significant development in genetic analysis, allowing amplification of minute amounts of a specified gene sequence (U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,800,159; B. I. Eisenstein (1990) The New England Journal of Medicine 322: 178-182; G. Schochetman et al. (1988) The Journal of Infectious Diseases 158: 1154-1157). In this method, a pair of single stranded oligonucleotide primers, each complementary to sequences on opposite strands of the target DNA, are selected to encompass the target sequence to be amplified and define the two ends of the amplified stretch of DNA. After separating double stranded DNA and annealing the primers to the 3' end of the target sequence on each strand, two complementary second strands are synthesized by extension of the annealed primers using a DNA polymerase, ie. a new single strand of DNA is synthesized for each annealed primer. These newly synthesized DNA's, as well as the original DNA sequence, can then be used for a second cycle of primer annealing and DNA synthesis. Accordingly, the desired target DNA sequence is amplified geometrically with each repetition of the cycle. Typically, within a few hours a target DNA sequence can be amplified 100,000 fold, particularly when automated methods are used to perform the cyclic reactions.
The polymerase chain reaction can also be used to specifically amplify only those target sequences which are expressed, ie., those which are transcribed. To do so, mRNA is isolated and cDNA is made from the RNA using reverse transcriptase. The cDNA, which represents the expressed genes, is then used as target DNA in the PCR amplification reaction.
Because of its high sensitivity and specificity, PCR has been successfully used as a means for identifying microorganisms and viruses in the diagnosis of infectious disease (B. I. Eisenstein (1990) J. Infectious Diseases 161: 595-602; L. Shih et al. (1990) J. Medical Virology 30: 159-162; A. R. Lifson et al. (1990) J. Infectious Diseases 161: 436-439; M. M. Anceschi et al. (1990) J. Virological Methods 28: 59-66). PCR amplification has been used to detect changes in expression of the dTMP synthase gene (ie., changes in the level of mRNA) associated with drug resistance in human tumors (K. J. Scanlon (1989) J. Clinical Laboratory Analysis 3: 323-329; M. Kashani-Sabet et al. (1988) Cancer Research 48: 5775-5778). PCR has also been used to analyze point mutations in HIV-1 reverse transcriptase which confer resistance to AZT (B. A. Larder et al. (1989) Science 246: 1155-1158) and point mutations in the dihydrofolate reductase-thymidylate synthase gene associated with pyrimethamine resistance in Plasmodium falciparum (A. F. Cowman et al. (1988) PNAS 85: 9109-9113; J. W. Zolg et al. (1989) Molecular and Biochemical Parasitology 36: 253-262; M. Tanaka et al. (1990) Molecular and Biochemical Parasitology 39: 127-134; J. W. Zolg et al. (1990) Molecular and Biochemical Parasitology 39: 257-266).
The above publications utilize the known PCR amplification method, in which a single pair of primers is used in each PCR reaction to amplify a target sequence representing a single gene. This method is usually adequate for research studies in which information about a particular gene is sought. However, it is not practical for detection of resistance to a drug in a clinical setting because resistance to a single drug can potentially involve any of several genetic elements corresponding to the multiple mechanisms of resistance to the drug. A clinical laboratory must also screen each clinical specimen for resistance to many drugs, each of which can be mediated by multiple genetic mechanisms. In such a setting, performing separate, sequential, PCR reactions (e.g., as disclosed in U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159) to detect each genetic element associated with resistance would be an expensive and time consuming procedure. The advantages of speed and simplicity associated with PCR would be lost by performing such individual assays on the scale required for drug resistance screening in a clinical laboratory.
The present invention improves upon several significant shortcomings of prior art drug sensitivity screening methods, namely 1) the unsatisfactory length of time involved in obtaining a result, 2) the fact that resistant microorganisms must comprise a significant portion of the microorganisms in order to be detected, and 3) the need to stop drug therapy to perform drug resistance screening. The inventive method utilizes simultaneous amplification of genetic elements specifically associated with drug resistance, which reduces expense and provides rapid screening. Multiple primer pairs, each specific for a genetic element associated with a particular mechanism of resistance to a drug, provide the basis for simultaneous amplification of the desired genetic elements. The inventive methods can be used to 1) identify, in a single simultaneous screening, the presence of potential resistance to a drug which is mediated by multiple genetic elements, or 2) to simultaneously screen for the presence of potential resistance to multiple drugs.
The particular advantages of the inventive method reside in the fact that it is not necessary to isolate or identify a microorganism prior to screening for drug resistance. Generally, for purposes of making a rapid, informed decision regarding selection of a drug for treatment of an infection, such specific information is not immediately required. It is usually of primary importance to know only whether a drug of choice is likely to be therapeutically effective, regardless of the etiology of the infection. The methods of simultaneous amplification of specific genetic elements of the present invention are capable of providing such information in a short period of time, and therefore allow more rapid initiation of therapy.